Project Summary Atheroprone flow-induced endothelial dysfunction, characterized by enhanced inflammatory response, is an early vascular event leading to the focal distribution of atherosclerosis. Despite the causality between atheroprone flow pattern and endothelial dysfunction, key molecular events linking mechanical stimuli to the pro-inflammatory phenotype of vascular endothelial cells (ECs) and the consequent susceptibility to atherogenesis remain poorly understood. We recently demonstrated that atheroprone flow activates sterol regulatory element binding protein 2 (SREBP2) in ECs, which induces the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome through transcriptional induction (i.e., Signal 1) and higher-order assembly (i.e., Signal 2). Such unresolved EC innate immune response leads to endothelial dysfunction and ensuing atherosclerosis. We have also identified that atheroprone flow induces TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA). Newly performed experiments indicate that SREBP2 transcriptionally upregulates TIFA and inflammasome components (Signal 1), and that phosphorylated TIFA Threonine-9 (TIFA pT9) oligomerizes TIFA leading to the higher-order assembly of NLRP3 inflammasome (Signal 2). These observations provide the foundation for the guiding hypothesis that atheroprone flow, through transcriptional induction and post-translational modification of TIFA, activates NLRP3 inflammasome in the endothelium, which contributes to atherosclerosis susceptibility. Three Specific Aims are proposed to test this hypothesis. Specific Aim 1 will elucidate the molecular mechanism by which atheroprone flow primes the induction of the NLRP3 inflammasome through Signal 1. In vitro flow channel experiments with oscillatory shear stress (OS) simulating atheroprone flow will be used to elucidate the transcriptional cascade of SREBP2-TIFA-NLRP3 inflammasome that respond to atheroprone flow. Specific Aim 2 will delineate the molecular basis by which atheroprone flow activates the NLRP3 inflammasome via Signal 2. Specifically, mechanobiology and FRET-based bioimaging will be used to investigate the role of TIFA pT9 in the higher-order assembly of NLRP3 inflammasome in ECs in response to atheroprone flow. Specific Aim 3 will investigate the role of atheroprone flow-activated SREBP2-TIFA-NLRP3 inflammasome in endothelial dysfunction, leading to atherosclerosis in vivo. We will examine endothelial function, vascular inflammation, and atherosclerosis in EC-SREBP2-/-, TIFA-/-, and TIFA T9A knock-in mice with or without an ApoE-/- background. The proposed experimental approaches that combine vascular, mechanobiological, biosensor, and animal experiments will systemically analyze the mechano and molecular basis of atheroprone flow-induced innate immune response in ECs and its functional relevance to atherosclerosis susceptibility.